Picking untilted/tilted particle pairs in EMAN2
The selection of untilted/tilted particle pairs from pairs of micrographs is a critical step in Random Conical Tilt (RCT) reconstruction and validation via tilt pairs http://www.ncbi.nlm.nih.gov/pubmed/14568533. The program e2RCTboxer.py implements this in EMAN2, and, having a similar look and feel, is based on e2boxer.py.
To run the program type:
e2RCTboxer.py <untilted micrograph> <tilted micrograph> options
Where options are:
--boxsize The starting boxsize for particle selection
- --slow Enables realtime update of particle position in the particles widget upon particle dragging in the mainwindow (I reccomend not using this)
Upon launching e2RCTboxer.py you will see four windows appear. Two main windows containing the untilted and tilted micrographs, a particle image window, which will contain the selected particles, and a control GUI. In the control GUI, main tab, there are two boxing tools, listed in a combobox, a Manual tool and a Pair Picker tool. The Manual picker allows the user to pick untilted/tilted particle pairs by alternately picking between the two boxes. For example, a untilted particle is selected, then a tilted particle, then a untilted, etc. When a particle is selected its index number listed in the upper left hand corner of the box. This number will be the same in both the untilted and tilted micrograph windows allowing the user to easily find pairs. In addition the untilted and tilted particle pairs are drawn to the particle image window, with the number listed in the lower left hand corner, and the type of particle (untilted or tilted) listed just above it. When a user deletes a particle the particle pair partner is also deleted in both the main windows and the particle image window. After all boxes have been selected, 'Write Output' should be pressed to write the particles to the database.
In addition to the main window tab, there is the Processor tab, which enables filtering of both micrographs using a EMAN2 filter, and the Filter tab, which enables a 3x3, 5x5 or 7x7 convolution kernel to be applied to both micrographs. Using either of these methods allows contrast enhancement for easier particle picking.
To enhance ease of use, the user can move to the Pair Picker mode, where once a minimum of three particle pairs are selected, a particle pair partner is picked once either the tilted or untilted particle is picked. To implement this, the program solves the linear transformation between the untilted and tilted micrograph. From this the computed tilt angle between the untilted and tilted micrograph, the tilt axis, and the gamma angle are computed. For more information see: http://www.ncbi.nlm.nih.gov/pubmed/19374019 and http://www.ncbi.nlm.nih.gov/pubmed/3058896.
The Pair Picker mode has the following options:
- Update box positions: When this is checked each time a new particle pair is selected and the linear transform recomputed, the tilted particle positions are recomputed.
- Center opposite box position: When checked, the particle pair partner is centered when its opposite is selected
- Min pairs for xform: The minimum number of particle pairs required for transform computation. At least three are needed.
- Computed tilt angle: The computed tilt angle between the untilted and tilted micrograph
- Computed tilt axis(Y): The tilt axis
- Gamma angle: See the papers listed above
- Mask type: Once the transform is known the linear transform of the micrograph outline can be drawn on the opposite micrograph to highlight the common area between them.
- Update Boxes: When pressed recomputes the box positions based on the current linear transform
- Center Boxes: When pressed centers the boxes so that the particle center is in the box center
- Clear: When pressed clears all the boxes
Strategies for success
- DO NOT use ice as fiducial markers. Ice sits atop the specimen and moves upon tilting and/or additional irradiation, hence your transform computation will be rubbish.
- DO use particles(best option) or vitrious ice embedded items as fiducial markers
- Initially pick particle pairs close to each other and then move outward